Univeral calibration system and method for a high performance, low volume, non-contact liquid dispensing apparatus

ABSTRACT

A universal calibration apparatus and method to estimate the dispense output from a low volume, non-contact, liquid dispensing systems that may be applied for every hardware configuration (e.g., tube length, orifice diameter, tip design, etc), reagent solution property and environmental condition. This same calibration technique is applied to calibrate or tune these non-contact liquid dispensing systems to dispense desired volumes (in the range of about 0.050 μL to 50 μL), irrespective of the hardware configuration or the solution properties. That is, the calibration technique is not dependent on any variables, but the result (the actual dispense volume) is dependant on the variable mentioned. By actuating selected pulse widths, and measuring the resulting volume, a Calibration Profile can be generated correlating the liquid volume dispensed from the orifice to the respective pulse width of the dispensing valve thereof through calibration points.. In particular, one is selected to deliver a first volume of liquid that is less than a lower base pulse width correlating to the lowest volume of the selected range of volumes of liquid, while a second pulse width is selected to deliver a second volume of liquid dispensed that is greater than an upper ceiling pulse width correlating to the highest volume of the selected range of volumes of liquid. Intermediary pulse widths are also applied, each selected to deliver a different, spaced-apart, respective intermediary low volumes of liquid dispensed from the dispensing orifice between the first volume and the second volume. Thus, applying the Calibration Profile, the pulse widths correlating to the one or more targeted discrete volumes for liquid dispensing can be extrapolated.

RELATED APPLICATION DATA

[0001] The present application claims priority under 35 U.S.C. §119 toU.S. Provisional Application Serial No. 60/351,858 (Attorney Docket No.INVDP004P), naming Johnson et al. inventors, and filed Jan. 25, 2002,and entitled METHODS FOR HIGH-PERFORMANCE, LOW-VOLUME DISPENSING, theentirety of which is incorporated herein by reference in its entiretyfor all purposes.

TECHNICAL FIELD

[0002] The present invention relates to methods and apparatus for liquidhandling, and more particularly, relates to methods and apparatus fornon-contact, high performance, relatively low volume liquid dispensing.

BACKGROUND ART

[0003] Advances in Life Sciences, particularly in genomics andproteomics, have greatly increased the potential number of reactions andanalyses that must be performed by the biotechnology and pharmaceuticalindustries. An estimated 30 million tests are required to screen atypical pharmaceutical company's compound library against targetreceptors. The typical number of tests will increase dramatically asinformation is gleaned from the sequencing of the human genome. To meetthese increasing throughput demands in an economically feasible manner,miniaturization of tests is imperative.

[0004] Technological advances are enabling the demonstration and use ofmicroscale chemical/biochemical reactions for performing various typesof analyses. Implementation of these reactions at such smaller scalesoffer economies that are unmatched by conventional approaches. Reducedvolumes can lower costs by an order of magnitude but conventionalliquid-handling devices fail at the required volumes. Parallelimplementation provides even greater advantages as demonstrated by theuse of high-density plates for screening and high-density MALDI-TOFplates for mass spectrometry analyses of proteins. The rate-limitinghardware is low volume liquid transfer technology that is robust andscalable for compounds of interest. With growing demand, the developmentof fluid handling devices adept at manipulating sub-microliter volumesof multiple reagents is needed.

[0005] Current systems for handling liquid reagents often employ a “pickand place” technique where a liquid reagent sample from a source plate,usually a microtiter plate, is picked up and placed into anotherreservoir known as the target plate. This technique is often applied forreplicating plates, where scale reduction between the source and thetarget plates are beneficially realized. Typically, an appropriatevolume is aspirated from a source plate and deposited to a target siteon a multiple target plate. In this arrangement, reduced sample volumesand sample spacing are required for higher degrees of miniaturization.These liquid handling systems can broadly be categorized into two liquiddispensing types: contact liquid dispensing devices and non-contactliquid dispensing devices.

[0006] One such type of contact liquid handling is capillary contactdispensing where physical contact is necessary for fluid transfer ofliquid reagents. By way of example, applying a thin, elongated pin tool,the tip of which is dipped into a liquid reagent sample in the sourceplate, and then maneuvered into physical contact with a substratesurface at the target site of the target plate for deposit of the liquidreagent sample thereon. Through capillary action, a certain amount ofliquid will adhere to the tip, and can then be transferred to the targetsite upon contact.

[0007] This approach, however, is inherently volumetrically inaccuratesince the amount of fluid adhered to the pin tool surface can vary witheach cycle. Moreover, due to “wicking” of the drops, relatively smalldispensing volumes, on the order of picoliters, cannot be repetitivelyattained with the sufficient accuracy required for scaled-down, highthroughput screening assays when delivering on dry surfaces. Further, toestimate the delivery volume, several physical properties and parametersmust be considered. These include the surface tension of the liquidreagent, the hydraulic state of the substrate surface, the affinity forthe substrate surface of the reagent fluid, the affinity for the pintool surface of the reagent fluid, the momentum of the delivery contact,, and the application of biochemical coatings on the substrate surfacejust to name a few. Another problem associated with this capillarycontact dispensing technique is that it is more vulnerable toinadvertent cross-contamination of the tool tip and target sites,especially when manipulating multiple reagents and the target sitedensity is high. Further, fragile biochemical coatings are oftenemployed on the surface of the test sites that can be easily damaged bythe tips of the pin tools during depository contact therebetween.

[0008] Regarding non-contact type liquid dispensing systems, liquiddispensing is performed without any physical contact between thedispensing device and the targeted substrate surface. Typically thesesystems include positive displacement, syringe-based liquid handlers,piezoelectric dispensers and solenoid-based dispensers, each technologyof which affords their own advantages and disadvantages.Piezoelectric-based systems, for example, are capable of accuratedelivery of low volume liquid handling tasks on the order of picoliters.Further, these devices are used with positional-accurate motion controlplatforms that enables increased test site array density.

[0009] While this approach is capable of accurate reagent delivery oflow volumes on the order of picoliters, one problem associated withthese systems is that dedicated or fixed sample reservoirs are requiredwhich are directly fluidly coupled to the dispense orifices of thepiezoelectric head. The application of this non-contact technique,however, is labor intensive when sub-microliter volumes of multiplereagents are required. Moreover, volumetric precision, at picoliterlevels are in part due to small dispensing orifice diameters that aresubject to frequent plugging. The scalability of these systems is alsoreduced since the small diameter of the orifice significantly limits thevolume dispense per pulse.

[0010] Solenoid-based actuation for non-contact liquid dispensing, onthe other hand, tend to be significantly more versatile and scalablecompared to the piezoelectric-based liquid dispenser systems. Usingconventional aspiration techniques to draw liquid reagent sample into aflow path or communication passageway (e.g., of a tube) of the system,relatively larger volumes or replicate smaller volumes can be dispensedwith high precision by the solenoid.

[0011] One problem associated with these designs, however, is that thesolenoid base actuator must be positioned in-line with the dispense flowpath. Accordingly, the flow of drawn reagent sample through thecomponents of the dispensing actuator can cause detrimental stiction.Ultimately, volumetric delivery imprecision results, as well aprematurely reducing the life of the dispensing actuator.

[0012] To address this problem, other compatible system fluids(typically filtered de-ionized water) are applied upstream from theaspirated liquid reagents to eliminate contact of the reagent with thedispensing actuator. This bi-fluid delivery approach has provensuccessful for dispensing a wide range of repetitive dispensing volumes.However, the aspiration of large overfill volumes is required due todispersion or dilution effect at the liquid interface between thesample\reagent and the system fluid. This especially holds true withrepetitive liquid dispensing where the repetitive actuation of thesolenoids causes increased agitation at the fluid interface. As shown inthe chart of FIG. 1 (illustrating the measured concentration of thedispensed reagent sample versus the dispense sequence), the measuredconcentration of the liquid reagent sample significantly degrades afteraround the 50^(th) to the 60^(th) discharge, although the volumetricaccuracy remains constant.

[0013] Accordingly, a scaleable, non-contact, liquid handling system andmethod is desired that provides repetitive, low volume, non-contactliquid dispensing without the degradation in liquid sample\reagentconcentration, and with volumetric precision ranging from microliters tonanoliters to

DISCLOSURE OF INVENTION

[0014] The present invention provides a universal calibration apparatusand technique to estimate the dispense output from for these low volume,non-contact, liquid dispensing systems that may be applied for everyhardware configuration (e.g., tube length, orifice diameter, tip design,etc), reagent solution property and environmental condition. This samecalibration technique is applied to calibrate or tune these non-contactliquid dispensing systems to dispense desired volumes (in the range ofabout 0.050 μL to 50 μL), irrespective of the hardware configuration orthe solution properties. That is, the calibration technique is notdependent on any variables, but the result (the actual dispense volume)is dependant on the variable mentioned.

[0015] Accordingly, this calibration technique must be performed forevery hardware configuration, and for every reagent liquid that will bedispensed from that particular hardware configuration. Briefly, in thiscalibration technique, the dispensing valve is precisely actuated afirst pulse width selected to deliver a first volume of liquid dispensedfrom the dispensing orifice that is less than a lower base pulse widthcorrelating to the lowest volume of the selected range of volumes ofliquid. The dispensing valve is again precisely actuated for a secondpulse width selected to deliver a second volume of liquid dispensed fromthe dispensing orifice that is greater than an upper ceiling pulse widthcorrelating to the highest volume of the selected range of volumes ofliquid. The dispensing valve is again precisely actuated for at leastthree different, spaced-apart, intermediary pulse widths, each selectedto deliver a different, spaced-apart, respective intermediary lowvolumes of liquid dispensed from the dispensing orifice between thefirst volume and the second volume. The first volume, the second volumeand the intermediary volumes are accurately measured. Briefly, althougha gravimetic and spectrophotometric techniques are preferred, anyaccurate volume measuring technique can be applied.

[0016] Using these measured volumes, and corresponding pulse widths, aCalibration Profile is constructed correlating the liquid volumedispensed from the orifice to the respective pulse width of thedispensing valve thereof through calibration points. Applyingcurve-fitting techniques the remaining Calibration Profile isinterpolated. Further, applying the Calibration Profile, the pulsewidths correlating to the one or more targeted discrete volumes forliquid dispensing can be extrapolated.

[0017] In yet another aspect of the present invention, a flow sensor isincluded to assess the liquid flow performance for dispensing liquidthrough a relatively small diameter dispensing orifice fluidly coupledto a communication passageway of a precision, low volume, liquidhandling system. The sensor includes an beam emitter that emits anoptical beam, from a position outboard from one side of the dispensingorifice, along an optical path extending substantially laterally acrossand downstream from the dispensing orifice of the liquid handling systemprior to dispensing liquid from the dispensing orifice. The sensorincludes a receiver that continuously senses the optical beam along theoptical path, from a position outboard from an opposite side of thedispensing orifice. When the dispensing liquid is flowed through thecommunication passageway which is generally sufficient to eject at leasta drop of dispensing liquid from the dispensing orifice, and across theoptical path of the optical beam, the drop is either detected of notdetected. If the drop is detected, the sensor indicates a flow conditionof the dispensing fluid through the dispensing orifice, and if the dropis not detected, a non-flow condition of the dispensing fluid throughthe dispensing orifice. In the latter case, a plugging problem may bedetected.

[0018] As the drop passes in from of the beam, the received optical beamis reduced in intensity by the receiver. Thus, the sensor detects achange or pause in the sensing of the optical beam.

[0019] The emitting device may be provided by a laser diode emitting theoptical beam. The receiving component must then detect wavelengthemitted by the diode. The sensitivity of the receiving component isadjustable so that the drop is more “visible” to the receivingcomponent. This may be performed by decreasing the intensity of theoptical beam received by the receiving component. A diffuser may beemployed to reduce the intensity.

[0020] In another embodiment, the sensor may be applied in amulti-channel liquid dispensing device where a plurality of relativelysmall diameter dispensing orifices are aligned in a substantially lineararray. In this arrangement, the sensor includes an emitter of an opticalbeam, from a position outboard from one side of the linear array of thedispensing orifices, along an optical path extending substantially alonga longitudinal axis of the linear array, and substantially laterallyacross and downstream from each dispensing orifice of the liquidhandling system prior to dispensing liquid from any one of thedispensing orifice. The sensor includes a receiving component thatcontinuously senses the optical beam along the optical path, from aposition outboard from an opposite side of the linear array of thedispensing orifices. As the dispensing liquid is flowed through arespective communication passageway with a pressure generally sufficientto eject at least a drop of dispensing liquid from one of the dispensingorifices, and across the optical path of the optical beam. If the dropis detected, the sensor indicates a flow condition of the dispensingfluid through the one dispensing orifice, and if the drop is notdetected, a non-flow condition of the dispensing fluid through the onedispensing orifice. This sequence must be sequentially repeating foreach of the remaining dispensing orifices to assess the operational flowcondition for the entire array of dispensing orifices.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The assembly of the present invention has other objects andfeatures of advantage which will be more readily apparent from thefollowing description of the best mode of carrying out the invention andthe appended claims, when taken in conjunction with the accompanyingdrawing, in which:

[0022]FIG. 1 is a chart illustrating the performance (measured inreagent concentration) of a sequence of 100 nl reagent dispensesutilizing a non-contact liquid dispensing system without the applicationof an in-line air gap.

[0023]FIG. 2 is a schematic diagram of the non-contact liquid dispensingsystem incorporating an in-line air gap constructed in accordance withthe present invention.

[0024]FIG. 3 is a schematic diagram of a multiple channel, non-contact,liquid dispensing system that incorporates the in-line air gap inaccordance with the present invention.

[0025]FIG. 4 is an alternative schematic diagram of FIG. 3.

[0026]FIG. 5 is a chart illustrating the performance (measured inreagent concentration) of a sequence of 100 nl reagent dispensesutilizing the non-contact liquid dispensing system with the applicationof an in-line air gap, in accordance with the present invention.

[0027]FIG. 6 is a top perspective view of a X-Y-Z “Pick and Place”mechanism utilized in combination with the present invention.

[0028]FIG. 7 is a Table of one specific set of frequencies utilized inthe “purge” routine in accordance with the present invention.

[0029]FIG. 8 is an enlarged, fragmentary bottom plan view, incross-section, of an orifice plug detection assembly constructed inaccordance with the present invention, and mounted to an array ofnozzles.

[0030]FIG. 9 is a fragmentary side elevation view, in cross-section, ofthe orifice plug detection assembly of FIG. 8.

[0031]FIG. 10 is a graph of a Calibration Profile for dispense volumesranging from 0.1 microliter to 1.0 microliter constructed in accordancewith the calibration technique of the present invention.

[0032]FIG. 11 is a Table of one specific set of measured calibrationpoints selected to construct the Calibration Profile of FIG. 10.

[0033]FIG. 12 is a graph of a Non-Linear Dispense Profile illustratingtransient and static flow through the dispensing valve correlating tothe open valve time (i.e., the pulse width).

[0034]FIG. 13 is a Table of data relating to three specific ranges ofdispensing volumes, including measured calibration points selected toconstruct the Calibration Profiles of FIGS. 14-18.

[0035]FIG. 14 is a graph of a Calibration Profile for dispense volumesranging from 1.0 microliter to 10.0 microliters constructed inaccordance with the calibration technique of the present invention.

[0036]FIG. 15 is a graph of a Calibration Profile for dispense volumesranging from 10.0 microliters to 40.0 microliters constructed inaccordance with the calibration technique of the present invention.

[0037]FIG. 16 is a graph of a Tri-Range Composite Calibration Profilefor the Calibration Profiles of FIGS. 10, 14 and 15.

[0038]FIG. 17 is a graph of a Dual-Range Composite Calibration Profilefor the Calibration Profiles of FIGS. 10 and 14.

[0039]FIG. 18 is a graph of a Dual-Range Composite Calibration Profilefor the Calibration Profiles of FIGS. 14 and 15.

[0040]FIG. 19 is a Table of data relating to three specific ranges ofdispensing volumes, including data for selecting the intermediarymeasured calibration points between the lower and upper base pulsewidths.

BEST MODE OF CARRYING OUT THE INVENTION

[0041] While the present invention will be described with reference to afew specific embodiments, the description is illustrative of theinvention and is not to be construed as limiting the invention. Variousmodifications to the present invention can be made to the preferredembodiments by those skilled in the art without departing from the truespirit and scope of the invention as defined by the appended claims. Itwill be noted here that for a better understanding, like components aredesignated by like reference numerals throughout the various figures.

[0042] Referring now to FIG. 2, a non-contact liquid handling method andsystem, generally designated 20, is provided which is capable of preciselow volume, liquid dispensing onto a target or destination substratesurface 21. Broadly, in one specific embodiment, the liquid handlingsystem 20 includes a pressure subsystem 22, a fluid aspiration (input)subsystem 23, a fluid dispensing (output) subsystem 25 and a fluidswitching subsystem 26. More particularly, the pressure subsystem 22 ofthe liquid handling system 20 includes a pressurized system fluidreservoir 27 independently fluidly coupled, via fluid pressure lines 28,30, to a fluid aspiration source 31 of the fluid aspiration (input)subsystem 23, and to a fluid dispensing source 32 of the fluiddispensing (output) subsystem 25. In turn, these sources 31, 32 areindependently fluidly coupled to the fluid switching subsystem 26 thatprovides fluid communication, via an elongated fluid communicationpassageway 33, to a dispensing orifice 35. In one configuration, thisorifice 35 is located at the distal end of a dispensing nozzle 36 thatenables both fluid aspiration and dispensing therefrom.

[0043] Further, as best shown in FIGS. 2 and 3, the fluid flow path ofthe system 20 is selectably switchable, via a switching valve 37 of theswitching subsystem 26, between a first fluid path A, by way of theaspiration source 31, and a second fluid path B, by way of thedispensing source 32. Thus, the first fluid path A extends from thesystem fluid reservoir 27 to the dispensing orifice 35, by way of theaspiration source 31, to aspirate fluids into the communicationpassageway, while the second fluid path B extends from the system fluidreservoir 27 to the dispensing orifice 35, by way of the dispensingsource 32, to dispense fluids from the communication passageway.Essentially, the first fluid path A controls metering of the fluid inputinto the dispensing orifice, while the second fluid path B controlsmetering of the fluid from the dispensing orifice.

[0044] Similar to other solenoid-based, non-contact, liquid handlingsystems, as will be described in greater detail below, the nozzle 36 andorifice 35 are arranged to aspirate a targeted dispensing liquid (e.g.,a liquid reagent sample) into the communication passageway 33 from areagent/sample source or source plate 38, as well as aspirate the liquidreagent sample therefrom. Unlike the current techniques, however, thepresent invention enables repetitive, precision low volume, non-contactliquid dispensing on the order of nanoliters without any concentrationdegradation of the sample due to the dispersion or dilution effect bythe system fluid 40 at the liquid interface.

[0045] Referring now to FIG. 2, this is accomplished by aspirating anair gap 41 into the communication passageway, via the dispensing orifice35, prior to aspiration of the liquid reagent sample therein. This airgap must extend continuously across the transverse cross-sectionaldimension of the communication passageway to completely separate thedistal end of the contained system fluid 40 from contact with theproximal end of the liquid reagent sample slug 42 drawn into thecommunication passageway. Since the integrity of this trapped air gap 41is maintained between the two opposed fluids for the duration of anyaspiration and dispensing operation, dispersion or dilution effects atthe interface between the liquid reagent sample 42 and the system fluid40 are substantially eliminated. That is, the air gap 41 must remainsubstantially intact and not fragment as the air gap reciprocate alongthe communication passageway 33.

[0046] Comparing the above-mentioned chart of FIG. 1, applying asolenoid-based liquid handling system without an in-line air gap withthe chart of FIG. 5, applying the same solenoid-based liquid handlingsystem with an in-line air gap, each repetitively delivering consecutive(i.e., about 350 dispenses), 100 nanoliter quantities of liquid, theperformance benefits for concentration stability are clearlyillustrated. Briefly, these charts illustrate the measured concentrationof the dispensed liquid reagent (in Relative Florescence Units (RFJ))versus the dispense sequence. In the chart of FIG. 1, the measuredconcentration of the liquid sample dispense significantly degrades afterthe 50^(th)-60^(th) consecutive dispense due to the dispersion ordilution effect with the system fluid at the liquid interface. Incomparison, the inclusion of an air gap 41 to separate the fluidssignificantly reduces the concentration degradation (FIG. 5) across theentire dispense sequence.

[0047] However, in order to provide precision, non-contact liquiddispensing at repetitive, substantially low volumes ranging fromnanoliters to microliters, it has been found that too large of an airgap separating the liquids is detrimental to performance. For example, a2 uL air gap can be used for replicate dispenses of 1 uL, but is toolarge for dispensing 100 nL. When too large a volume air gap isaspirated between the system fluid and the reagent fluid, compression ofthe in-line gas (air gap) can occur during a liquid dispense procedure.This compression of the air gap 41 unpredictably affects the efficiencyof the pressure pulse as it traverses the air gap 41. In effect, avariable pressure drop occurs at the air gap/liquid interface instead ofat the nozzle orifice.. It is therefore difficult to reproduce andcontrol the magnitude of the pressure pulse across the dispense orificedue to such added compliance in the system. Accordingly, the requisiteprecision and reproducibility for low volume, non-contact, liquiddispensing as low as the nanoliter range can only be achieved with airgaps in the range of 0.05-5 uL. By way of example, such compression hasbeen observed for aspirated air gap volumes greater than or equal toabout 10.0 microliters in pressure lines having a 0.020″-0.035″ innerdiameter for the communication passage. The air gap reacts to thepressure pulse much like a spring compressing then expanding.

[0048] In accordance with one aspect of the present invention, it hasbeen found that aspirating smaller volumes of in-line gas significantlyimprove the volumetric dispensing precision and reproducibility notcapable with volumes greater than about 5.0 microliters. Applying asingle, continuous air gap in the range of about 250 nanoliters to about2.0 microliters, extending across the transverse cross sectionaldimension of the communication passageway, precision low volume,non-contact, liquid dispensing can be delivered from these non-contact,liquid dispensing devices while maintaining sufficient separation of theliquids at the interface to minimize dispersion and dilution by thesystem fluid.

[0049] Using high-speed photography, it has been observed that thesediscrete air gaps essentially behave as substantially incompressiblefluids, unlike air gaps larger than about 10 microliters for the givendiameters. Accordingly, as the pressure pulse propagates down thecommunication passageway (originating from the back pressure and theopening and closing of the dispensing actuator as will be describedbelow), the pressure pulse can traverse this discrete air gap interfacewithout significant loss of energy due to compliance. This enablesgreater control of the pressure pulse across the dispensing orifice 35at the nozzle 36 for repetitive, precise, low volume, non-contact liquiddispensing in the picoliter to microliter range.

[0050] Referring back to FIGS. 3 and 4, and as viewed in FIG. 6, thenon-contact, liquid handling system 20 will now be described in greaterdetail. In the preferred form, the liquid handling system 20 is aprovided by multi-channel liquid dispensing device capable ofsimultaneous, multiple reagent dispensing from multiple nozzles 36.Similar to our UNIVERSAL NON-CONTACT LIQUID HANDLING PERIPHERALAPPARATUS, which is the subject of U.S. patent application Ser. No.10/237,916, filed Sep. 6, 2002, the entirety of which is incorporatedherein by reference for all purposes, a remote fluidic module 43 isincluded that houses the fluid aspiration (input) subsystem 23, thefluid dispensing (output) subsystem 25 and the fluid switching subsystem26. The pressure subsystem 22 includes the pressurized system fluidreservoir 27, having a ⅛″ pressure line 45 coupled to a digital pressureregulator 46 of the fluidic module 43. In turn, the pressure regulator46 is fluidly coupled to a pressure source (not shown) having a maximumpressure of preferably about 50 psi. The preferred back pressureregulated by the digital pressure regulator is in the range of about 2psi to about 15 psi, and more preferably retained in the range of about8 psi. It will be appreciated, however, that the selected back pressureis dependent upon several factors, as will be discussed, including theparameters of the dispensing actuators, the dispensing orifice diameterand the nozzle design (creating backpressure at the orifice), thequantity to be liquid dispensed, and the viscosity of the liquid sampleand system fluid. Accordingly, during operation, a constant backpressure is maintained in both the first fluid path A and the secondfluid path B. It is this constant back pressure, in combination with theactuation of the dispensing actuators 32 (e.g., a solenoid dispensingvalve), that creates the necessary pressure pulse to eject the drop ofliquid reagent from the nozzle orifice 35.

[0051] Briefly, the pressure head at the dispensing valve 32 is createdby system pressuring gas acting upon the system fluid (preferablyfiltered de-ionized water) at the system fluid reservoir 27. Preferably,a pressurizing gas is selected that scavenges gas bubbles in the systemfluid and suppresses in-gassing and is not soluble in the system fluid.In-gassing into the system fluid can result in bubble or micro-bubbleformation, and poor dispense performance caused by rapid solenoidactuation. Micro-bubbles in the system fluid, as differentiated from airgaps, will degrade dispensing performance due to pressure drops in thesystem. Micro-bubbles are uncontrolled and not intentionally introducedinto the communication path, whereas the air gap is precisely controlledand intentional. For instance, as the pressure pulse propagates throughthe communication passageway 33, the cumulative effect of thesemicro-bubbles increases compliance that in turn, decreases the pressuredrop across the dispensing orifice 35. A potential pit-fall of theout-gassing of the pressurizing gas is that air can become trappedwithin the previously primed solenoid. The trapped air will then createa situation where the solenoid will dispense a greater volume than itdid when it was fully primed. The solenoid will open, allowing systemfluid to pass valve. This will compress the trapped air within thesolenoid. The system fluid has now displaced the compressed air that istrapped within the solenoid valve, As the valve closed the trapped airexpands and pushes the system fluid that compress the air gap out of thevalves.

[0052] One such pressurizing gas that is particularly advantageous ishelium, when using an aqueous pressurized system fluid. This inert gasscavenges air (Nitrogen) gas bubbles in the system fluid and suppressesthe formation of additional bubbles. The solubility of Helium in aqueoussolutions is also less than that of air, nitrogen or argon. Accordingly,the application of helium enables the use of non-degassed, aqueoussystem fluids.

[0053] Incidentally, filtered de-ionized water is the most typical andbenign liquid chosen as the system fluid. It will be appreciated,however, that other fluids and aqueous reagents can be substituted,depending upon the application as long as the surface tension of thatfluid enables the formation of the air gap between the system fluid andthe sample reagent.

[0054] Applying a {fraction (1/16)}″ID PFA pressure line 30, the secondfluid path B is fluidly coupled to an eight (8) port manifold device 47(FIG. 3) that distributes the system fluid into eight independentchannels. More or less ports and corresponding channels may be applied,of course. In turn, each port of the manifold device 47, via pressurelines 48, is fluidly coupled to a corresponding solenoid dispensingvalve 32 which in turn is fluidly coupled to a hybrid valve manifold 49that channels fluids to the switching valve 37. These dispensing valves,which as mentioned are preferably solenoid-based, and deliver a meteredpressure pulse using a pressure ranging from about 6.9(10)³ N/m² (1 psi)to about 138(10)³ N/m² (20 psi), and having a duration ranging fromabout 200 μs to about 10 seconds. Preferably, these dispensing valves 32are provided by conventional ink-jet style printing valves or pumpsdesigned for drop-on-demand printing. For instance, the Lee Company ofEssex, Conn. manufactures a solenoid-based ink-jet valve (Model No.INKX0502600AB) that is suitable for use with the present invention.

[0055] As best viewed in FIG. 3, the output of each solenoid dispensingvalve 32 is directly mounted to the multiple path, hybrid valve manifold49. The internal switching valve 37 is preferably provided by a rotaryshear face valve to effect precision positioning when switching betweenthe fluid aspiration system and the fluid dispensing system. Thisswitching subsystem is the subject of U.S. patent application Ser. No.09/689,548, filed Oct. 11, 2000, and entitled HYBRID VALVE APPARATUS ANDMETHOD FOR LIQUID HANDLING, the entirety of which is incorporated hereinby reference for all purposes. Briefly, through the rotary shear facevalve/hybrid manifold, the selected solenoid dispensing valves can befluidly coupled to a selected nozzle 36.

[0056] Regarding the first fluid path A, an ⅙″ID PFA pressure line 28extends from the system fluid reservoir 27 to the aspiration source 31which is preferably, an eight (8) channel syringe drive, driven by asingle motor drive. This multi-channel syringe drive simultaneouslydistributes and aspirates the system fluid into eight independentchannels. This external analytical metering device, such as asyringe-based pump or a diaphragm pump, is capable of precision meteredaspiration of small fluid quantities in the range of at least 250nanoliters to about 2.0 microliters into the communication passageway ofeach associated pressure line 50 through the corresponding dispensingorifice 35. Typical of these aspiration devices is Model # 2009Dprovided by Innovadyne Technologies, Inc., Santa Rosa, Calif. Similar tothe solenoid dispensing valve 32, the output of each analyticalsyringe-based drive 31 is fluidly coupled to the associated inputs ofthe hybrid switching valve 37, via respective 22 gauge FEP pressurelines 44.

[0057] The output lines 50 from the hybrid valve 37 to the correspondingnozzles 36 provide the corresponding communication passageways 33 ofeach independent channel. Each line is preferably provided by TEFLON®(e.g. PFA\FEP) pressure tubing having sufficient flexibility to enableprecision positioning of the associated nozzle 36 above either thesample source for aspiration of the sample reagent into thecorresponding communication passageway, or the destination substratesurface or microtiter plate for high performance low volume dispensingthereof.

[0058] Referring now to FIGS. 3 and 6, the Universal Non-Contact LiquidHandling Peripheral 20 incorporates a “pick and place” mechanism 51having a base 52 and an automated motion control component 53 tocollectively manipulate precision positioning of the nozzles 36, as aunit. Briefly, the motion control component 53 preferably employs aconventional three-axis X-Y-Z Cartesian system, and precision track orrail hardware to position the nozzles along the X-axis and Y-axis (i.e.,vertically above the targeted test sites 54 of the micro titer plate21), and along the Z-axis. In one specific arrangement, the dispensinghead provides eight (8) dispensing nozzles 36 aligned in a linear arrayhaving dispensing ends equally spaced-apart by a distance conforming tothe spacing of the wells or test sites 54 of the microtiter plate 21.Other conventional positioning mechanism may also be applied, such asthose having one placement component movable in the Z-axis directionabove another plate component movable in the X-axis and Y-axisdirection, the respective nozzles can be maneuvered above the sourceplate and into the targeted sample reservoir.

[0059] Further, an operation interface component 55 provides standaloneor remote operation of all subsystems (e.g., the fluidics module 43 andthe “pick and place” mechanism 51). More particularly, the interfacecomponent 55 operates and integrates the fluid control and motioncontrol components. Incorporated in this chassis are all of the printedcircuit boards, connectors, and firmware required to operate theinstrument. Software may reside on a host computer independent of theinterface component.

[0060] The hybrid valve apparatus and the non-contact liquid dispensingperipheral are adaptable for printing arrays wherein the distancebetween adjacent test sites 54, or test site pitch, is in the range ofabout 1 micron (μm) to about 10,000 microns (μm). Thus, the presentinvention is particularly suitable for transferring chemical orbiochemical samples or reagents from the sample source plate 38 havingan array of reservoir wells 56 (e.g., a conventional microtiter platewith 96 or 384 wells) to an array of higher-density test sites 54 (e.g.,a 1536-well microtiter plate), or for fabrication of a chip-basedbiological sensor (commonly referred to as a “microarray”) used forperforming gene expression or other screening experiments. The sourceplates are generally used in sample analysis protocols, and aretypically provided by plastic plates defining uniformly spaceddepressions or wells (i.e., test sites 54) that contain the fluiddispensing therein. These commercially available rectangular platesfurther typically include eight rows of twelve microwells that providean industry-standard ninety-six microwell plate, such as plate 21 shownin FIG. 6. Other conventional sizes include sixteen rows of twenty-fourmicrowells that provide three hundred eighty-four microwells.

[0061] Referring back to the TEFLON (PFA\FEP) pressure lines 50, theseelongated, chemically inert lines are selected to perform functionsother than merely providing the respective fluid communicationpassageway 33 between the hybrid valve outlet and the respective nozzlesorifice 35. For example, to further reduce in-gassing, the lines can beselected to be substantially chemically inert to biological fluids andcommonly used solvents, such as DMSO, THF, alcohols, aldehydes, ketones,halogenated hydrocarbons, aliphatic hydrocarbons, acids and bases usedin the life sciences and diagnostic fields. These pressure lines mustalso be sufficiently flexible to enable X-Y-Z positioning of the nozzles36, which are mounted to the X-Y-Z “pick and place” mechanism 51 (FIG.6). Further, the remote placement of the solenoid actuators 32, relativethe nozzles 36, allows for enhanced flexibility in designing thedispensing systems. That is, several factors are considered whenselecting the overall length of the pressure lines 50, as well as theinternal diameter of the communication passageway.

[0062] Although the length of the pressure line 50 is a factor indetermining the sum volume of the communication passageway 33, to bediscussed later, it has been found desirable to position the in-line airgap 41 sufficiently downstream from the solenoid dispensing actuator 32in order to maintain the integrity of the air gap 41 across thetransverse cross-sectional dimension of the communication passageway.Depending upon the back pressure at the system fluid reservoir 27,should the air gap 41 be positioned too close to the solenoid dispensingactuator 32, the initial shock or impact from the pressure pulsepropagating down the communication passageway 33 may be sufficient tofragment the air gap. Since the volume of the air gap 41 is very minute(i.e., about 250 nanoliters to about 2.0 microliters), dilution anddispersion of the opposed liquids at this interface is likely with anyfragmentation.

[0063] Accordingly, by lengthening the pressure lines 50 by a sufficientamount in addition to the targeted cumulative volume of liquid reagentsample aspirated into the communication passageway 33, the air gap 41can be positioned sufficiently downstream from the dispensing actuator32 so that the system fluid volume 40 in the passageway 33 partiallyisolates and cushions the impact of the pressure pulse on the air gap41. Moreover, it is believed that the flexibility of the pressure lines50 themselves help dampen the initial impact of the pressure pulsethrough motion absorption as the flexible pressure lines moves andflexes upon initial entrance of the pressure pulse in the communicationpassageway 33. By way of example, for a flexible pressure line having aninternal diameter in the range of about a 0.020″-0.035″ ID, a backpressure in the range of about 2 psi to about 15 psi, and an air gap 41of about 250 nanoliters to about 2.0 microliters, the air gap 41 ispreferably positioned downstream from the entrance into thecommunication passageway in the range of at least 1.0 inch to about 3.0inches.

[0064] Another consideration when selecting the pressure lines 50 andcorresponding fluid handling components is maintaining the integrity ofthe minute air gap 41 as it reciprocates in the communication passageway33. This is primarily performed by providing relative smooth walltransitions within the communication passageway 33. Such smoothtransitions are critical to preventing fracture of the air gap 41 as itmoves through the communication passageway. This holds especially trueat any component interface, such as between the pressure line 50 and thenozzle 36. Abrupt transitions, such as stepped transition from a largerdiameter to a smaller diameter or protruding objects from the interiorwalls, may impact the integrity of the air gap as it passes by.Accordingly, a significant effort is afforded to match the properties ofthe components to smooth all transitions, especially componentinterfaces. Operations such as electrochemical polishing of theStainless Steel probes and beveling of the Stainless Steel tube thatconnect the nozzle to the fluid line can minimize dispersion effectscaused by stepped transitions. Controlling the dimensions of all thefluid lines and channels helps to enhance performance and reduceimprecision.

[0065] Other factors influencing fluid dispensing include the interiordiameter of the communication passageway, the back pressure created bythe nozzle design (e.g., straight or angled passageway) and orificediameter, the viscosity of the liquid reagent fluid to name a few. Instill other considerations, as indicated, the length of the pressureline 50 can be tailored to the targeted dispense application. Generally,in accordance with the present invention, the smaller the quantity offluid to be dispensed for these non-contact fluid dispensing systems 20,the shorter the length requirements of the pressure lines, whereas, thegreater the quantity of low volume fluid dispensing, the longer thelength requirement, outside of the mere volume considerations of thecommunication passageway.

[0066] By way of example, for a smaller quantity of liquid dispensed(e.g., 50 nl), the length of the pressure line should be preferablyreduced to maintain the requisite pressure drop across the nozzleorifice 35 that is necessary to eject the drop cleanly in these minutevolumes. This is due in part because the solenoid dispensing valves 32are required to operate within an optimum back pressure range to assureproper performance. Too low a back pressure will not be sufficient tocleanly eject the drop from the nozzle orifice, while too high a backpressure will prevent proper operation of the valve (e.g., preventingopening of the valve at all). The optimum back pressure range for mostsolenoid dispensing valves 32 in this category is from 4 psi to about 15psi.

[0067] However, to effect smaller dispensing quantities, smaller pulsewidths are required, resulting in an overall lower pressure. Too long alength pressure line will likely cause too small of a pressure dropacross the orifice 35 since such a drop is a function of the pressureline length. That is, the longer the pressure line length, the greaterthe pressure drop due to the incremental pressure loss caused byfriction between the interior wall and the fluid. Although the trappedair and dead volume within the solenoid valve 32 and communicationpassageway 33 are preferably purged, as will be detailed in the purgeroutine described below, to reduce compliance within the system, thereare still pressure losses due to the friction of the fluid with thewalls of the pressure line. Too small a pressure drop across the orificecauses reduced ejection volumes, given the same pulse width and fluidreservoir back pressure. Consequently, residual sample fluid maybuild-up at the orifice 35 during subsequent ejections until oneparticular ejection carries this build-up in the ejected drop,significantly increasing the dispensing volume. This of course resultsin volumetric imprecision and variance. For longer pressure linelengths, in comparison, greater quantities of low volume dispensing aredelivered accurately by adjusting the back pressure or pulse width toachieve the requisite pressure drop for clean ejection of droplets.

[0068] As an example to this aspect of the present invention, to effectabout a 50 nl fluid dispense, with a back pressure nominally at about 8psi, the length of the pressure line, having an interior diameter ofabout 0.028 inch nominal, should be in the range of about 2.0 inches toabout 12 inches. In another example, for a 200 nl fluid dispense, usingthe same system pressure , the length of the pressure line 50 should beabout 2.0 inches to about 118 inches (3 meters). Generally, with longertube lengths, either the pulse width or the back pressure must beincreased, relative to the shorter tube length, to deliver an equivalentamount of fluid. This is provided that the viscosity of the fluidremains unchanged.

[0069] Depending upon the predetermined volume to be aspirated intocommunication passage, which incidentally is predicated upon thecumulative volume of repetitive dispensing from the nozzle (to bediscussed), the length of the pressure line 50 and the inner diameterthereof may be determined. Using long-tube, remote dispensing with fromabout 5.0 inches to about 120.0 inches of about 0.020 inch ID to about0.035 inch ID tubing between the nozzle orifice 35 and the solenoidbased actuator 32 (FIG. 2, volumes of 25 nanoliters to 70 microliterscan be dispensed with a dispense performance of less than 5% RelativeStandard Deviation (RSD). By way of example, applying the presentinventive method of aspirating minute air gaps between the fluids, suchlength fluid communication lines 50 having a 0.028″ nominal internaldiameter, can yield about two-thousand (2000) to about forty-fourthousand (44,000), 25 nanoliter volume dispenses each havingsubstantially equal concentrations (comparing FIG. 1 (the systemapplication without a minute air gap) to FIG. 5 (the system applicationwith an air gap)). To illustrate scalability, this approach can alsoyield about one (1) to about twenty-five (25), 40 microliter volumedispenses as well. In other examples, using a single 500 μl aspiratedvolume, about forty-eight hundred (4800), 100 nanoliter dispenses toabout four-hundred eighty (480), 1 microliter dispenses withsubstantially equal concentrations can be attained having an RSD lessthan 5%. Variations include systems where the tubing diameter variesform 0.010″ ID to 0.040″ ID; tubing length greater than 120″ with RSDless than 10%.

[0070] Application of the present inventive technique will now be morefully described. Referring to FIG. 2, prior to aspirating the minute airgap 41 into the communication passageway, the system fluid 40 in eachcommunication line 50 must be maneuvered to the end of the correspondingdispensing orifice 35 regardless of which hydraulic state the system isin (e.g., dry, partially dry or wet hydraulic state). Applying thebackpressure of the system fluid reservoir, this may be performed eitherthrough the solenoid dispensing valve 32 (second fluid path B) or thesyringe-based aspiration actuator 31 (first fluid path A), or both.

[0071] Regarding the second fluid path B, the switching valve 37 isoriented to enable fluid communication between the solenoid dispensingvalves 32 and the corresponding communication passageways 33. Thesolenoid dispensing valve 32 can then be operated from a closedcondition, preventing or closing fluid communication between thecommunication passageways and the corresponding solenoid dispensingvalves 32, to an opened condition, enabling or opening fluidcommunication between the communication passageways and thecorresponding solenoid dispensing valves 32. The constant back pressureof the pressurized system fluid reservoir 27 is then applied to thesystem fluid for flow thereof through solenoid and into thecommunication passageway 33. This is performed until the system fluid isdispensed from the corresponding dispensing orifices 35 in asubstantially constant and bubble free manner. A trapped gas purgeroutine may then be applied which will be described in greater detailbelow.

[0072] Similarly, regarding the first fluid path A, the switching valve37 is oriented to enable fluid communication between the syringe-basedaspiration actuator 31 and the corresponding communication passageways33. A three-way valve in the syringe drive can be opened to enable fluidcommunication between the communication passageways and the system fluidreservoir. Again, the constant back pressure of the system fluidreservoir 27 or the priming action of the syringe drive can be appliedto flow the system fluid through the channels of the hybrid valve andthe corresponding communication passageways 33 until exiting the systemin a manner substantially constant and bubble free.

[0073] Once the system fluid is satisfactorily moved all the way to theend each dispensing orifice 35 and any trapped gas is deemed purged fromtheir respective communication passageway and solenoid dispensing valve32, as will be described, the nozzles 36 may be positioned verticallyover the respective wells of the source plate 38, via the “pick andplace” mechanism 51, prior to aspiration of the respective reagentsample. With the hybrid valve 37 positioned to enable fluid aspiration,each corresponding precision analytical syringe drive 31 is operated toaccurately meter air, as a unit, into the communication passageway 33,via dispensing orifice 35. In accordance with the present invention,this separating volume ranging from about 150 nl to about 5 μl, and morepreferably about 250 nl to about 2 μl.

[0074] Applying an X-Y-Z “pick and place” mechanism 51, such as thatUniversal Non-Contact Liquid Handling Peripheral 20 above-mentioned, therespective nozzles 36 can be maneuvered into the targeted samplereservoir. Subsequently, actuating the corresponding precisionanalytical syringe drive 31, a single continuous slug of the reagentfluid sample is drawn into communication passageway. Preferably, theminute air gap 41 is maintained within the communication passageway 33of the associated tube, and is not drawn into the hybrid valve 37. Whilethe air gap 41 may be positioned upstream from the hybrid valve 37, itis preferable to retain the air gap downstream from the hybrid valve 37,and maintain the minimum downstream distance from the solenoiddispensing valve by merely lengthening the corresponding pressure lines50. As indicated above, the length and ID of the lines are selected as afunction of the volume predetermined to be aspirated into thecommunication passage.

[0075] The hybrid valve 37 is operated to switch the respectivecommunication passageways 33 from communication with the correspondinganalytical syringe drives 31 to the corresponding solenoid dispensingvalves 32 remotely located on the fluidics module. Applying theabove-mentioned techniques and the “pick and place” mechanism should be51, the nozzles 36 are repositioned above their destination test sites54. The solenoid-based actuators 32 are precisely actuated between theclosed condition to the opened condition to control the pulse width(i.e., the length of time the valve is opened) to determine the volumeof the drop ejected from the corresponding nozzle orifice. As mentionedabove, and using the calibration techniques to be discussed below, thepulse width corresponding to volume liquid reagent ejected from theorifice is a function of many factors, including the viscosity of theliquid reagent sample, the length of the lines 50, the ID of the line,the back pressure of the system fluid reservoir, the resulting backpressure across the nozzle orifice which is a function of the nozzledesign. For example, to effectively dispense a solution such as 30%Glycerol/water, a higher back pressure is required, a longer pulse widthis required, and slower aspirate speeds must be used relative toperforming a dispense of a less-viscous solution such as Hexane. Theability to empirically calibrate a variety of fluids, through the use offluorescent labeling or gravimetric measuring, enables the developmentof a matrix of compound classes that can be referenced by the instrumentto use as offsets from a pre-determined calibration.

[0076] In accordance with another aspect of the present invention, amethod has been developed to purge trapped air within the solenoid basedactuators 32 when the system fluid is initially flowed through theactuators and the communication passageways from a dry air-filled stateto a wet hydraulic state. As mentioned, the purging of such trappedgases is imperative for precise , non-contact, liquid dispensing atthese low volumes. Each trapped air bubble or micro-bubble in thesolenoid itself, and those adhered to the walls of the communicationpassageway 33, as compared to the minute air gap 41 traversely extendingthe entire communication passageway, micro-dampen the pressure pulsepropagating down the communication passageway. The collective influenceof this compliance in the system, however, results in a significantsystem pressure loss resulting in an ineffective pressure drop acrossthe nozzle orifice 35 that can reduce volumetric precision. Also, thespring like contraction and expansion of the bubbles causes imprecisedispensing.

[0077] Using repetitive fixed pulse solenoid actuations, a “buzz”routine has been developed to dislodge the trapped gases in thedispensing actuator and corresponding communication passageway 33 of thepressure line 50 that are ultimately purged out from the nozzle orifice.Applying fast actuations, opening and closing the dispensing valve athigh frequency bands, together with the back pressure of the systemfluid reservoir, the routine effectively purges or expels bubbles or airtrapped in the solenoid valves. Since relatively high frequenciesactuations in the range of 1 Hz to about 1700 Hz are applied, codedfirmware is thus required to properly perform the routine. Morepreferably, the frequency range of about 10 Hz to about 420 Hz areutilized.

[0078] It has been found particularly effective to vary the solenoidactuation frequency to assure complete purging of the trapped bubbles.Depending upon the amount of trapped gas within the solenoid, thedifferent frequencies of the actuation are effective in dislodging thegas within the solenoid. The consequently reduced compliance of thepurged solenoids, has great impact on the performance of multiplesolenoid systems, greatly improving dispense precision and stability ofmulti-channel systems. For example, one (1) to twenty-five (25) discretefrequency bands can be applied in a random frequency range from about 10Hz to about 420 Hz. In another specific embodiment, as shown in theTable I of FIG. 7, a fixed pulse ramped frequency increase sweep fromabout 1 Hz to about 420 Hz over one (1) to fifteen (15) discrete,equally-spaced frequencies. Thus, it is the different discrete frequencybands that has been found effective, although certain delivery patternsmay be even more effective. By way of example, one routine may include aramped frequency decrease sweep, with denser frequency spacings at thehigher or lower frequencies. Briefly, the actual actuation frequency inthe fourth column of Table I of FIG. 7 is not linear at the highernBuzzCount rate since the execution of the each actuation command takesabout 200 us. At the relatively low frequencies, this is not much of afactor but it becomes a factor with higher repetitions.

[0079] In a multiple channel system, such as in our UniversalNon-Contact Liquid Handling Peripheral Apparatus, above-mentioned,simultaneous purging of the communication passageways 33 can occurthrough simultaneous actuation of the corresponding solenoid dispensingvalves 32 coupled thereto. However, not all solenoid dispensing valvesand their associated communication passageways will be purged equallyand at the same rate. According, a technique has been developed todetermine the quality of the purge in all pressure lines aftercompletion of the “buzz” routine.

[0080] This is performed by generating a pressure pulse through eachcorresponding solenoid dispensing valve, each of which has substantiallythe same pulse width. Essentially, an attempt is made to dispensesubstantially equal volumes of system fluid independently from eachpressure line. The liquid dispensed from each respective nozzle orificeis collected to determine the dispensed volume. Conventional measuringtechniques can be employed, such as by weighing, spectrophotometric, oroptical methods. For example, applying a back pressure of approximately8 psi, operating the solenoid dispensing valves 32 to generate atwenty-eight thousand (28,000) μs pulse width should typically yieldabout a thousand (1000) nanoliters of system fluid.

[0081] Applying these measured volumes of dispensed liquid, the meanvariance is calculated. For any of the pressure lines 50 that delivereda fluid amount having a measured volume that exceeds the mean varianceby an amount greater than a predetermined figure, trapped gas may stillremain in the solenoid actuator and/or the communication passageway.Thus, this continued compliance is the cause of such measured differencefrom the mean variance. In one specific embodiment, the predeterminedfigure is in the range of about 3% to about 7% difference from thecalculated mean variance, and more preferably about 5%. Variance is thepercent difference of the average for a single tip compare to the meanof the dispenses for all of the tips, as set forth herein:

%Variance=((Tip mean-Mean of all tips)/Mean of all tips)*100.

[0082] To address these differences, the purge routine for thoseparticular lines or the entire set of lines is repeated, and thedispense sequence and volume measurement is repeated. In fact, thisentire procedure is repeated until each line delivers fluid quantitiesthat differ from the mean variance within the predetermined figure. Oncethe variance is within specifications all pressure lines 50 and theirassociated solenoid dispensing valves have been properly purged oftrapped bubbles and are hydraulically intact.

[0083] In still another aspect of the present invention, an apparatusand technique has been developed to monitor the flow or lack of flowthrough the dispensing orifices 35 in each respective nozzle 36. Sincethe nozzle or dispensing orifices 35 are relatively small, in a range ofpreferably about 50 microns to about 250 microns, plugged dispensingorifices are an inherent problem in these low volume dispense systems.Thus, a plug detection sensor assembly, generally designated 70, isapplied to detect “plugging” of the orifice 35. As best viewed in FIGS.9 and 10, the sensor assembly 70 is mounted at the tip of the nozzles 36which are carried by the automated motion control component 53 of the“pick and place” mechanism 51. Preferably, the sensor assembly isprovided by an optical “through beam” sensor having an optical beamemitter component 71, emitting an optical beam, and a receivingcomponent 72, sensing the optical beam. Alternative optical devicevariations may be employed however.

[0084] By placing and aligning the components of the sensor assembly 70just downstream from the dispensing orifice 35, any flow of liquiddispensed from the dispensing orifice 35 will impair the transmission ofthe beam of light from the optical emitter component 71 to the receivingcomponent 72. Accordingly, proper ejection of dispensed liquid from thenozzle orifice 35 will impair passage the beam of light, indicating thatthe integrity of the dispensing orifice has not be compromised, and thatproper operation can commence. Moreover, since these low volume liquiddispensing systems typically eject micro-droplets of liquid from thedispensing orifice 35, as opposed to a continuous stream of liquid, thebreakage from continuous receipt of the light beams are on the order ofmilliseconds. Once the droplet has passed, the light beam is againreceived and detected by the receiving component 72. By way of example,for the passage of a about 50 nanoliter liquid dispense, applying asystem fluid back pressure of approximately 8 psi, the breakage of thebeam is only on the order about 2-3 ms. In contrast, should therespective nozzle orifice 35 be completely clogged, the breakage of thebeam will not occur, indicating a potential problem. In moresophisticated operations, where larger volumes are to be dispensed fromthe nozzle orifice 35, partial clogged orifices may be detected. Forinstance, for a predetermined dispensing volume of a known liquid, andknown hardware and parameters (e.g., system fluid back pressure, orificediameter, exit velocity, etc.), the time required to break the beam maybe known or estimated. However, should the break in the beam be of aperiod significantly less than the known or estimated period, although abreak does occur, a partially clogged orifice may be detected In oneconfiguration, each nozzle 36 may include a dedicated sensorcorresponding to each nozzle orifice 35. Such an arrangement would ofcourse be substantially more costly. In another specific embodiment, asillustrated in FIG. 10, the sensor components 71, 72 of the sensorassembly 70 are preferably placed in-line, longitudinally, with thearray of nozzles 36 so that only one sensor is required. In thisarrangement, the optical emitter component 71 is placed outboard fromone end of the nozzle array while the receiving component 72 is placedoutboard from the other opposite end of the array. Thus, the opticalemitter can emit a light pulse across the entire array (e.g., 6-12)nozzles orifices 35 such that the ejection of a drop of liquid from anyone of the orifices will impair detection of the light beam by thereceiving component 72.

[0085] In this approach, each individual channel must of course bemonitored independently when detecting flow or lack of flow through thecorresponding orifice 35. The firing and detection sequences can be inany order, as well as any clog or partial clog detections can bedetermined, controlled and operated using sensor hardware and associatedwith the software/firmware code, data acquisition, fault determination,algorithms and recovery protocols that respond to the acquired data.

[0086] The optical emitter component 71 of the optical sensor assembly70 is preferably provided by a laser diode or the like. The opticalreceiving component 72 is of course selected to detect light in thewavelength range transmitted by the diode. For example, the laser diodemay be selected to transmit light in the visible range which isbeneficial in that these are safe wavelengths, and use inexpensivecomponents. One such optical sensor suitable for this application is theSun-X Optical Sensor (Model No. FX-D1), manufactured by SUNX of Nagoya,Japan.

[0087] To facilitate detection of the light beam transmitted across thenozzles, it may be necessary to decrease the intensity of the light beamemitted by the diode. When the emitted light received by the receivingcomponent is too intense, the beams essentially pass through the drop,especially when substantially transparent, alluding detection.Decreasing the light intensity, in effect, yields an overall increase inthe detection sensitivity so that the ejected drops become more“visible” to the receiving component.

[0088] One technique applied to reduce the light beam intensity sensedby the receiving component 72 is to position a diffuser or filter 73 infront of the face of the receiving component. This is accomplished bycovering the face of the receiving component 72, and providing a smallaperture 74 that reduces the amount of light transmission through to thereceiving component. Preferably, the aperture is in the range of about0.005 inch to about 0.030 inch in diameter, and more preferably is about0.020 inch in diameter. Other arrangements that reduce the lightintensity received by the receiving component include optical filters.

[0089] The sensor assembly 70 includes a pair of brackets 75 a, 75 b(FIG. 10) that enable mounting to the motion control unit 53, relativethe dispensing orifices 35 of the nozzles 36. These mounting brackets 75a, 75 b are arranged to face the corresponding optical components 71, 72inwardly toward one another for transmission and receipt of the lightbeam. In one configuration, the transmission face of the optical emittercomponent 71 and the receiving face of the receiving component 72 arepreferably positioned in the range of about 0.25 inch to about 2.0inches downstream from the corresponding end nozzle orifice 35, and morepreferably about 0.75 inch downstream therefrom. Further, each componentface is preferably positioned in-line along the longitudinal axis of thearray of nozzle orifices 35, but laterally spaced outboard from thecorresponding end nozzle orifices about 0.25 inch to about 2.0 inches,and more preferably about 0.75 inch. These brackets 75 a, 75 b, may alsoprovide adjustments to enable fine tuning of the position of the sensorcomponents.

[0090] In another aspect of the present invention, a method is providedfor calibrating the volume dispensed from these low volume, non-contact,liquid dispensing systems 20 before application of the present inventivedispensing methods. As mentioned, these systems rely upon pressurizedfluids and micro-dispense (solenoid) valves to generate fluid flowthrough the communication pathways and ultimately the dispensingorifices 35. Unlike conventional syringe-based pump technologies, systemconfigurations, reagent fluid properties and environmental conditionssignificantly contribute to the flow output and the dispensed volumefrom the system, as will be discussed. Conventional syringe pumptechnologies, for instance, use mechanical drives to fill and empty asyringe. Generally, regardless of the fluid properties, system designand environmental conditions, the volume of fluid filled into anddispensed from the syringe in these systems is directly proportional tothe number of steps that the syringe drive is commanded to move.

[0091] Periodically, the syringe drive may be calibrated to evaluate theaccuracy and precision of these mechanical pumps. It is not possible toadjust these stepped drives to improve accuracy, instead the drive mustbe commanded to move a different number of steps other than thetheoretical number of steps to achieve accuracy. For example, this maybe determined by the following equation:

Volume (steps)=(Volume desired)(Total number of steps per fullstroke/syringe volume) (e.g. 750 Volume (steps)=250 uL(15000 steps/500uL)=7500 Steps).

[0092] The technology applied in solenoid-based dispensing valves 32 isvery different from that of the positive displacement syringe pump. Asindicated-above, the solenoid dispensing valves 32 and the pressurizedsystem fluid reservoir 27 of the liquid dispensing system 20 cooperateto perform liquid dispensing by actuating the dispensing valve fromclosed condition to the opened condition for various time periods todeliver different volumes of liquid reagent sample to the destinationsite. The volume of liquid dispensed from the dispensing valves isproportional to the length of time that the valve is held open. Thedispensing volume from these systems is, thus, dependant upon severalfactors including: the time the valve is opened; the back pressure ofthe system fluid; the diameter of the dispensing orifice; and thedistance between the micro-dispense valve and the tip (i.e., thefriction between the fluid and the walls of the pressure lines).Accordingly, the numerous variables that are involved makemathematically calculating the dispense volume, based upon the length oftime of valve is opened, extremely laborious and difficult for such highprecision low volume liquid dispensing instruments.

[0093] A universal calibration technique has therefore been developed toestimate the dispense output from for these low volume, non-contact,liquid dispensing systems 20 that may be applied for every hardwareconfiguration (e.g., tube length, orifice diameter, tip design, etc),reagent solution property and environmental condition. This samecalibration technique can be applied to calibrate or tune thesenon-contact liquid dispensing systems to dispense desired volumes (inthe range of about 0.050 μL to 50 μL), irrespective of the hardwareconfiguration or the solution properties. Thus, while the implementationof this calibration technique is not dependant on these variableparameters, such as valve open time, system fluid back pressure, orificediameter or tube length, etc., the Calibration Profile generated fromthese measured quantities is dependant upon such above-mentionedparameters. That is, the calibration technique is not dependent on anyvariables, but the result (the actual dispense volume) is dependant onthe variable mentioned.

[0094] Accordingly, this calibration technique must be performed forevery hardware configuration, and for every reagent liquid that will bedispensed from that particular hardware configuration. Briefly, when thesame hardware configuration and liquid reagent sample is to be dispensedfrom each orifice of these multi-channel liquid dispensing systems, thiscalibration technique may be performed systematically, and then averagedfor each channel. In other instances, however, where channels may havedifferent hardware configurations, and where different liquid reagentsamples of varying dispense volumes may be dispensed, then thiscalibration technique may be performed per each individual channel.Other environmental factors such as temperature may also affect theCalibration Profile. To insure proper performance it is desirable that acalibration be performed at the site where the instrument will be used.

[0095] In accordance with the present inventive calibration technique, aCalibration Profile is to be generated graphing the dispense volume as afunction of the pulse width (i.e., the open time of the solenoid valve32). An example of this is the Calibration Profile of FIG. 10 togetherwith the Table II of FIG. 11, illustrating the dispense volume (e.g.,nanoliter) vs. the pulse width (e.g., microseconds). For a dispensevolume range of about 0.1 microliter to about 1 microliter, tencalibration points have been selected to generate the CalibrationProfile. However, it is possible to use less calibration points or morecalibration points. Disregarding practicality, the greater the number ofpoints, the greater the accuracy of the Calibration Profile that isgenerated. Typically, once the target range of volume to be dispensed isdetermined for a particular dispensing session or procedure, calibrationpoints should at the very least be selected to be below the lower basepulse width and above the upper ceiling pulse width. Table II of FIG.11, by way of example, shows the lower base pulse width of 3,600microseconds and the upper ceiling pulse width of 30,000 microseconds tobracket the range of 0.100 μL to 1.00 μL. Several intermediate pointsshould then be selected within the targeted volume range of liquiddispensing as well, one technique of which will be exemplified below.Other than through experience and educated estimates, the pulse widthsrequired to dispense liquid volumes less than the lower base width, andgreater than the upper ceiling pulse width for the targeted range ofvolumes are estimated. In the example of FIGS. 10 and 11, for a pressureline of about 51 inches in length, a dispensing fluid density of about0.9977735 g/mL, a back pressure of about 8.00 psig and an ambienttemperature of about 23° C., a pulse width of 3600 microseconds has beenselected to dispense a volume of the reagent liquid below the lower basevolume range of 0.1 microliter. Inputting a pulse width of 3600microseconds into the operation interface component 55 of the liquiddispensing system 20 essentially opens the solenoid valve for this timeperiod. Incidentally, these solenoid dispensing valves above-mentionedmay be precisely actuated open for periods as small as about 300microseconds. While the minimum spike actuation time at the spikevoltage of 24 Volts is 250 microseconds for these valves, 200microseconds actuations are attainable in combination with a crystalclock frequency of 20 microseconds.

[0096] The dispensed volume is collected, and then measured to determinethe actual volume dispensed. Briefly, as will be described in greaterdetail below, two low-volume measuring techniques are applied inconnection with the present invention. One low volume measuringtechnique involves weighing the dispensed fluid (i.e., gravimetrictechnique), while the other involves measuring the Relative FluorescentUnits (spectra-photometric technique) of the low dispensed volumes.There are many detection techniques such as absorbance, luminescence,and mass spectrometry that can be used. Differing detection techniquesallow a broad range of chemistries to calibrated. Regardless ofdetection technique, multiple replicate pulses at a single pulse widthare delivered, measuring the average dispensed volume. Such averagingreduces any errors in accuracy that would be caused by variances inprecision from each individual dispense. Moreover, for multi-channelapplications having identical hardware configurations, etc., asabove-indicated, systematic calibration can occur averaged by the numberof channels.

[0097] Once the average dispensed volume is measure and calculated,which for the initial pulse width of 3600 microseconds is about 0.097microliters, the calibration point along the Calibration Profile can beplotted. Essentially, the measured volume delivered can be correlated tothe open valve time (i.e., the pulse width). Applying more points, asillustrated in Table II of FIG. 11, the Calibration Profile of FIG. 10can be generated for the volume range of 0.1 microliter to about 1microliter for this particular hardware configuration. Once this isestablished, any dispensing volumes within this target volume dispensingrange may be delivered through interpolation techniques with accuracy inthe range of about −5% to about +5%. Further, through the operationinterface component 55 which incorporates the necessarysoftware/firmware code, data acquisition, fault determination,algorithms and recovery protocols that respond to the acquired data, thedelivery volumes can be automated.

[0098] For low volume, non-contact liquid dispensing in the very lowvolume ranges attainable through the application of solenoid dispensingvalves 32, where the valve openings (i.e., the pulse widths) arecontrolled in the (200 microseconds=0.2 milliseconds) microsecond range(i.e., 1×10⁻⁶ seconds), the selection of the calibration points iscritical. Generally, at commencement of flow through the solenoiddispensing valve 32, the flow velocity increases until a maximum flow isreached. In this range, the rate of change of the flow of reagent (i.e.,acceleration) through the valve is increasing and the flow is thus notsteady-state. Once maximum velocity is attained, the flow issubstantially not changing where the rate of change of the flow (i.e.,acceleration) is substantially zero. The dispense profile, thus, becomeslinear.

[0099] This is exemplified more clearly in the Dispense Profile graph ofFIG. 12, correlating the flow (nanoliters/millisecond) vs. the pulsewidth (microseconds). Viewing the Dispense Profile graph is perhaps moreintuitive than viewing the Calibration Profile of FIG. 10 where theinflection region or point can be seen where the flow becomessubstantially steady somewhere between about 8,000 microseconds to about10,000 microseconds, in this instance. Experience has shown that maximumflow velocity typically occurs approximately at dispensing volumes innear to 0.5 microliter for a wide variety of hardware configurations,solution properties and ambient conditions.

[0100] Due to the non-linear, non-steady-state, fully developed, laminarnature of flow until maximum flow velocity is reached, it is thistransient region that is more difficult to profile. Accordingly, themajority of the calibration points should be established within this(non-steady-state, fully developed, laminar) region. Thus, a greaternumber of pulse width selections should be allotted in this region whenconstructing the Calibration Profile (FIGS. 10 and 12). In contrast, inthe more linear region of the dispense profile where the flow issubstantially steady, fewer calibration points are required determinedto interpolate the Calibration Profile. It will be appreciated, however,that consideration of the transient flow is more imperative at very lowvolume dispensing (between about 0.10 microliter to about 1.00microliter), and at very short pulse widths (in the range of about 3,000microseconds to about 10,000 rmicroseconds). In contrast, when thetarget dispense volume is to occur where the flow velocity is at maximumvelocity, such transient flow need not be considered because the fluidis no longer accelerating.

[0101] When target delivery volumes span a substantially wide range ofvolumes (e.g., from about 0.1 microliter to about 50.0 microliters), thecalibration profiles may be separated into discrete narrowly definedvolume ranges (i.e., three or more Calibration Profiles traversing oroverlapping different volume ranges). Subsequently, these profiles canbe combined into one composite Calibration Profile. In Table III of FIG.13, for example, the volume ranges are separated into three differentvolume ranges: 0.1 microliters to 1.0 microliter, 1.0 microliter to 10.0microliters and 10.0 microliters to 40.0 microliters.

[0102] Applying the above-mentioned calibration technique, theCalibration Profiles were constructed for each different volume range.For the first volume range in the range of 0.1 microliters to 1.0microliter, the measured data of which is set forth in more detail inTable III of FIG. 13, the resulting Calibration Profile is shown in FIG.10. Similarly, for the second volume range of 1.0 microliter to 10.0microliters, the resulting Calibration Profile is shown in FIG. 14,while the third volume range of 10.0 microliters to 40.0 microlitersyields the resulting Calibration Profile of FIG. 15. Depending upon thetarget volume dispensing range, these Calibration Profiles can becombined to yield a wider range Calibration Profile. FIGS. 16-18,accordingly, yield Tri-Range and Dual-Range Calibration Profiles, thedata of which can be input into a software interface for automatedoperation. It will be appreciated that the interface need not begraphical. Further, while the profiles can be combined, the software andfirmware can be adapted to support an number of data points (ten areexemplified in this example, and were provided to show that thecalibration ranges overlap at 1 μL and 10 μL) in the final calibrationtable.

[0103] As previously mentioned, the initial pulse widths selected toprepare these Calibration Profiles are estimated using a lower basepulse width that will deliver a volume less that the lowest targetvolume for the calibrated range, and an uppermost ceiling pulse widththat will deliver a volume that is greater than the highest targetvolume in the calibrated range. This is exemplified in the second volumerange (i.e., 1.0 microliter to 10.0 microliters) of Table III of FIG.13, where the lower base pulse width and associated dispense volumeoverlap the upper ceiling pulse width and associated dispense volume ofthe first volume range (i.e., 0.1 microliter to 1.0 microliter).Similarly, the upper ceiling pulse width and associated dispense volumeof the second volume range overlap the lower base pulse width andassociated dispense volume of the third volume range (i.e., 10.0microliters to 40.0 microliters). In particular, in the second volumerange of 1.0 microliter to 10.0 microliters, a lower base pulse width of23,700 microseconds was selected which yielded a dispense volume ofabout 0.841 microliters. On the upper base end, a pulse width of 200,000microseconds was selected which yielded a dispense volume of about11.772 microliters.

[0104] In one technique to determine the intermediate pulses for themiddle of this Calibration Profile, the lower base pulse width isdivided into the upper ceiling pulse width to calculate the multiples ofthe base pulse width to the ceiling pulse width. The log of thisquotient is then calculated to determine the multiplier that is used tocalculate the pulse widths that will be used in the middle of theCalibration Profile.

[0105] As set forth below, and as exemplified in Table IV of FIG. 19,the data of which corresponds to that in Table III, the quotient of thesecond volume range is determined as follows:

200,000 μs/23,700 μs=8.438819.

[0106] Subsequently,Multiplier=Quotient^(1/(number of calibration points))Multiplier=1.267419.

[0107] A multiplier of 1.267419 is then calculated to determine theintermediary pulse widths. For example, 23,700 μs×1.267419=30,038 μs,while 30,038 μs×1.267419=38,071 μs, etc.

[0108] Using this curve-fitting technique of determining the pulsewidths of the Calibration Profiles, calibrations can be developed formany ranges and numbers of calibration points. This curve-fittingtechnique is beneficial for several reasons. First, this techniqueprovides the flexibility to calculate the pulse widths that are usedwithin a calibration range, rather than guessing at pulse widths thatwill bracket the target dispense volume. Guessing leads to the necessityto perform iterations of each calibration until the correct one isdetermined. Secondly, the dispense volume relative to the pulse widthappear to increase logarithmically. Therefore, selecting a lower basepulse width, and then a subsequent pulse width that is a logarithmicdeviation from the base pulse width, rather than a linear deviation,should yield improved accuracy when the calibration profile is used insoftware to select the dispense pulse. Lastly, this technique spaces thesubsequent pulse widths such that there are more points at the lower endof the curve and fewer points at the upper end of the curve (e.g., Seethe “Pulse Range” column in Table IV of FIG. 19). The time differencebetween point one and two is 956 μs, between point two and three 1210μs, between points three and four 1532 μs, and the difference betweenall subsequent pulses are increasing to greater length of time.

[0109] Briefly, regarding the “Periods Pulse” column of Table IV of FIG.19, the dispenser does not have the ability to dispense to theresolution of a single microsecond. The Xtal or Clock frequency is setto 20 microseconds. The instrument will receive a command to dispense at3600 microseconds. The firmware will then convert this time to periods(3600 μs/(20 us/period))=180 periods. The dispense will then dispensefor 180 periods of 20 microseconds. If the dispense time is notdivisible by twenty or the set clock frequency the firmware will roundthe number of dispensing periods to the lower integer. A calculatedpulse with of 4556 microseconds is converted to 227.8 periods with isthe rounded to 228 periods or 4560 microseconds.

[0110] As mentioned above, the actual measurement of the dispensedvolume from the dispensing orifices 35 can be determined using either agravimetric calculation or spectrophotometric calculation. These twomethods will be discussed briefly below.

[0111] With regard to the gravimetric calculation, this techniqueinvolves weighing of the dispensed fluid (i.e., gravimetric technique).By measuring the mass of fluid delivered after each pulse, the fluidvolume delivered can be easily calculated and can be correlated to anopen time of the respective solenoid valve. Typically, multiple pulsesat a single pulse width are delivered to a container. The container anddispensed liquids are weighed to determine the weight of the dispensedliquid once the container weight is removed. The reason that multiplepulses are used in the calibration technique is to reduce any error inaccuracy that would be caused by variances in precision from eachindividual dispense. Another technique, should the hardwareconfiguration for each channel be substantially similar, is tosimultaneously actuate each solenoid dispensing valve with identicalpulse widths, and then average the measured dispensing volumes for eachchannel. In this manner, a systematic, as opposed to individual,calibration may be performed.

[0112] The volume of fluid delivered with each pulse is determined bythe following equation:${{Volume}({nL})} = \frac{{{Density}\left( {g\text{/}{mL}} \right)}/\left( {{{total}\quad {{mass}(g)}} - {{tare}\quad {{mass}(g)}}} \right)*\left( {1 \times 10^{6}\quad {nL}\text{/}{mL}} \right)}{\left( {{Number}\quad {of}\quad {Pulses}} \right)*{\left( {{Number}\quad {of}\quad {Tips}} \right).}}$

[0113] As mentioned The pulse widths used to prepare these CalibrationProfiles are calculated by starting with a lower base pulse width thatwill deliver a volume less that the lowest target volume for thecalibrated range and an uppermost ceiling pulse width that will delivera volume that is greater than the highest target volume in thecalibrated range. The lower base pulse is then divided into the upperceiling pulse to calculate the multiples of the base pulse to theceiling pulse. The log of this quotient is then calculated to determinethe pulses that will be used in the middle of the Calibration Profile.Using this technique of determining the pulses of the CalibrationProfiles, calibrations can be developed for many ranges and numbers ofcalibration points. Different pulse widths are used for each calibrationpoint such that the total masses of all of the final volumes aresimilar. In this manner the scale is always measuring a similar mass.However, in order to maintain a statistically significant number ofpulses for calibration, each calibration point preferably applies aminimum of ten pulses.

[0114] Regarding the spectrophotometric volume calculation (orfluorescent calibration method), a technique is used where liquiddispensing occurs at several different pulse widths (i.e., valve opentime periods) into a microplate capable of optimal fluorescence. Theplate used in this method is usually black plate, due to the lowbackground. By measuring the fluorescence within each well and comparingthat fluorescence to a standard curve, a volume can be calculated. Oncethe volume is known then the relationship of volume and pulse width canbe plotted on an graph. Multiple pulses at a single pulse are deliveredto single well of a microtiter plate and the total fluorescence ismeasured in a fluorescent plate reader. The reason that multiple pulsewidths are used in the calibration technique is to reduce any error inaccuracy that would be caused by variances in precision from eachindividual dispense. The tables in the gravimetric section below showpulses used to calibrate the dispenser in three different volume ranges100 to 1,000 nL, 1,000 to 10,000 nL and 10,000 to 40,000 nL.

[0115] The sequence of events to calibrate with the fluorescent methodare outlined below:

[0116] Dispense in to a black microplate

[0117] Add diluent to the microplate

[0118] Shake the plate for one minute

[0119] Read the plate

[0120] Calculate volume from the fluorescent standard curve,calculations below.

[0121] Volume delivery calculation

[0122] Export calibration plate and dispense plate RFJ (relativefluorescence units) data to Excel

[0123] Calculate linear regression slope for the calibration plate

[0124] Convert RFU data on the dispense plate to concentration

[0125] Calculate the volume of dye delivered to each well

[0126] Concentration Dye Dispensed

[0127] y=Mx+b (slope of the linear regression)

[0128] Volume calculation—Two Methods

[0129] V₁=(C₂V₂)/C₁ use if V₂>>V₁

[0130] C₂=value obtained from linear regression

[0131] C₁=Stock solution concentration (˜500,000 nmol/L)

[0132] V₂=volume of diluent added

[0133] 100 uL=100,000 nL

[0134] 500 nL added to 100 uL˜Error of 0.5%

[0135] C₁V₁=C₂V₂ use if V₂˜V₁

[0136] C₁V₁=C₂(V₁+V_(dil))

[0137] C₁V₁=C₂V₁+C₂V_(dil)

[0138] C₁V₁−C₂V₁=C₂V_(dil)

[0139] V₁(C₁−C₂)=C₂V_(dil)

[0140] V₁=(C₂V_(dil))/(C₁−C₂)

[0141] C₂=value obtained from linear regression

[0142] C₁=Stock solution concentration (˜500,000 nmol/L)

[0143] V_(dil)=volume of diluent delivered to the well

[0144] Again, the pulse widths used to prepare these CalibrationProfiles are calculated by starting with a lower base pulse width thatwill deliver a volume less that the lowest target volume for thecalibrated range and an uppermost ceiling pulse width that will delivera volume that is greater than the highest target volume in thecalibrated range. The lower base pulse width is then divided into theupper ceiling pulse width to calculate the multiples of the base pulseto the ceiling pulse. The log of this quotient is then calculated todetermine the pulses that will be used in the middle of the CalibrationProfile. Different pulses are used for each calibration point such thatthe total masses of all of the final volumes are similar. In this mannerthe scale is always measuring a similar mass. This is even moreimportant when the calibration transitions to a fluorescent method forcalibration. Fluorescent microplate readers have linear ranges that aremuch narrower than analytical scales. However, in order to maintain astatistically significant number of pulses for calibration, eachcalibration point preferably applies a minimum of ten pulses.

[0145] Although only a few embodiments of the present inventions havebeen described in detail, it should be understood that the presentinventions may be embodied in many other specific forms withoutdeparting from the spirit or scope of the inventions.

What is claimed is:
 1. A method for calibrating a non-contact, liquiddispensing apparatus to enable substantially precise, low volume, liquiddispensing of one or more targeted discrete volumes of a selecteddispensing liquid within a selected range of volumes of liquid, thedispensing apparatus providing a communication passageway having aproximal end fluidly coupled to a fluid reservoir maintained at asubstantially constant positive pressure for ejection of the dispensingliquid out of an opposite end dispensing orifice of the communicationpassageway, the dispensing apparatus further including a precisionactuation dispensing valve in fluid communication with the communicationpassageway downstream from the fluid reservoir and adapted for rapidactuation between a closed condition, preventing flow of the pressurizedfluid through the actuation valve from the fluid reservoir to thedispensing orifice, and an opened condition, enabling flow of thepressurized fluid through the dispensing valve from the fluid reservoirto the dispensing orifice, the method comprising: (a) preciselyactuating the dispensing valve from the closed condition to the openedcondition and back to the closed condition for a first pulse widthselected to deliver a first volume of liquid dispensed from thedispensing orifice that is less than a lower base pulse widthcorrelating to the lowest volume of the selected range of volumes ofliquid; accurately determining the first volume of liquid dispensed; (b)precisely actuating the dispensing valve from the closed condition tothe opened condition and back to the closed condition for a second pulsewidth selected to deliver a second volume of liquid dispensed from thedispensing orifice that is greater than an upper base pulse widthcorrelating to the highest volume of the selected range of volumes ofliquid; accurately determining the second volume of liquid dispensed;(c) precisely actuating the dispensing valve from the closed conditionto the opened condition and back to the closed condition for at leastthree different, spaced-apart, intermediary pulse widths, each selectedto deliver a different, spaced-apart, respective intermediary lowvolumes of liquid dispensed from the dispensing orifice between thefirst volume and the second volume; accurately determining eachrespective intermediary volume of liquid dispensed; (d) forming aCalibration Profile correlating the liquid volume dispensed from theorifice to the respective pulse width of the dispensing valve thereofthrough calibration points determined from the first volume, the secondvolume, and the at least three intermediary volumes, and thecorresponding lower base pulse width, upper base pulse width and theintermediary pulse widths, and extrapolating the Calibration Profilesubstantially through the calibration points.
 2. The method according toclaim 1, further including: determining one or more target pulse widthsfrom the Calibration Profile correlating to the one or more targeteddiscrete volumes of liquid for application on the dispensing valve tosubstantially accurately dispense the one or more targeted discretevolumes of liquid from the dispensing orifice.
 3. The method accordingto claim 1, further including: estimating an inflection point along theCalibration Profile where transient flow through the dispensing valveoccurs in a transient flow region on one side of the inflection point,and static flow occurs in a static flow region on the other side of theinflection point; in the transient flow region, precisely actuating thedispensing valve from the closed condition to the opened condition andback to the closed condition for at least two different, spaced-apart,transient pulse widths, each selected to deliver a different,spaced-apart, respective transient volumes of liquid dispensed from thedispensing orifice. accurately determining each respective transientvolume of liquid dispensed; and further forming the Calibration Profilethrough the transient calibration points determined from the respectivetransient volumes, and the corresponding transient pulse widths, andfurther extrapolating the Calibration Profile substantially through thetransient calibration points.
 4. The method according to claim 2,further including: in the static flow region, precisely actuating thedispensing valve from the closed condition to the opened condition andback to the closed condition for at least one static pulse width todeliver a respective static volume of liquid dispensed between theinflection pulse width and the upper base pulse width; accuratelydetermining the static volume of liquid dispensed; and further formingthe Calibration Profile through an at least one static calibration pointdetermined from the at least one static volumes, and the correspondingstatic pulse widths, and further extrapolating the Calibration Profilesubstantially through the at least one static calibration point.
 5. Amethod for assessing the liquid flow performance for dispensing liquidthrough a relatively small diameter dispensing orifice fluidly coupledto a communication passageway of a precision, low volume, liquidhandling system, the method comprising: emitting an optical beam, from aposition outboard from one side of the dispensing orifice, along anoptical path extending substantially laterally across and downstreamfrom the dispensing orifice of the liquid handling system prior todispensing liquid from the dispensing orifice; continuously sensing theoptical beam along the optical path, from a position outboard from anopposite side of the dispensing orifice; flowing the dispensing liquidthrough the communication passageway generally sufficient to eject atleast a drop of dispensing liquid from the dispensing orifice, andacross the optical path of the optical beam, wherein; detecting the dropindicates a flow condition of the dispensing fluid through thedispensing orifice; and wherein not detecting the drop indicates anon-flow condition of the dispensing fluid through the dispensingorifice.
 6. The method according to claim 5, wherein the emitting of anoptical beam includes activating a laser diode emitting the opticalbeam.
 7. The method according to claim 6, wherein the continuouslysensing the optical beam includes detecting the optical beam through areceiving component of a sensor assembly configured to detect theoptical beam emitted from the diode
 8. The method according to claim 7,wherein the detecting the drop includes adjusting the sensitivity of thereceiving component so that the drop of dispensed liquid from thedispensing orifice is more “visible” to the receiving component.
 9. Themethod according to claim 8, wherein the adjusting the sensitivityintensity of the receiving component of the sensor assembly includesdecreasing the intensity of the optical beam received by the receivingcomponent.
 10. The method according to claim 9, wherein the decreasingthe intensity of the received optical beam is performed by positioning adiffuser in the optical path of the optical beam between the dispensingorifice and the receiving component of the sensor assembly.
 11. Themethod according to claim 5, wherein the detecting the drop includesidentifying a change in the continuous sensing of the optical beam, andnot detecting the drop includes not identifying a change in thecontinuous sensing of the optical beam.
 12. The method according toclaim 11, wherein the identifying a change includes detecting the pausein the continuous sensing of the optical beam, and the not identifying achange includes not detecting a pause in the continuous sensing of theoptical beam.
 13. A method for assessing the operational flow conditionfor dispensing liquids through a plurality of relatively small diameterdispensing orifices aligned in a substantially linear array and eachfluidly coupled to a respective communication passageway of a precision,low volume, liquid handling system, the method comprising: (a) emittingan optical beam, from a position outboard from one side of the lineararray of the dispensing orifices, along an optical path extendingsubstantially along a longitudinal axis of the linear array, andsubstantially laterally across and downstream from each dispensingorifice of the liquid handling system prior to dispensing liquid fromany one of the dispensing orifice; (b) continuously sensing the opticalbeam along the optical path, from a position outboard from an oppositeside of the linear array of the dispensing orifices; (c) flowing thedispensing liquid through a respective communication passagewaygenerally sufficient to eject at least a drop of dispensing liquid fromone of the dispensing orifices, and across the optical path of theoptical beam, wherein; (d) detecting the drop indicates a flow conditionof the dispensing fluid through the one dispensing orifice; and wherein(e) not detecting the drop indicates a non-flow condition of thedispensing fluid through the one dispensing orifice; and (f)sequentially repeating events (c)-(e) for the remaining dispensingorifices to assess the operational flow condition for the entire arrayof dispensing orifices.
 14. The method according to claim 13, whereinthe emitting of an optical beam includes activating a laser diodeemitting the optical beam.
 15. The method according to claim 14, whereinthe continuously sensing the optical beam includes detecting the opticalbeam through a receiving component of a sensor assembly configured todetect the optical beam emitted from the diode
 16. The method accordingto claim 13, wherein the detecting the drop includes adjusting thesensitivity of a receiving component of a sensor assembly so that therespective drop of dispensed liquid from the respective dispensingorifice is more “visible” to the receiving component.
 17. The methodaccording to claim 16, wherein the adjusting the sensitivity intensityof the receiving component of the sensor assembly includes decreasingthe intensity of the optical beam received by the receiving component.18. The method according to claim 17, wherein the decreasing theintensity of the received optical beam is performed by positioning adiffuser in the optical path of the optical beam between the dispensingorifice and the receiving component of the sensor assembly.
 19. Themethod according to claim 13, wherein the detecting the drop includesidentifying a change in the continuous sensing of the optical beam, andnot detecting the drop includes not identifying a change in thecontinuous sensing of the optical beam.
 20. The method according toclaim 19, wherein the identifying a change includes detecting the pausein the continuous sensing of the optical beam, and the not identifying achange includes not detecting a pause in the continuous sensing of theoptical beam.